CN115920991A - Micro-fluidic chip and method for sorting microorganisms - Google Patents
Micro-fluidic chip and method for sorting microorganisms Download PDFInfo
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Abstract
The invention discloses a micro-fluidic chip and a method for sorting microorganisms, belongs to the technical field of rapid aseptic microorganism detection, and particularly relates to a method for achieving the purpose of rapid aseptic detection of special biological product samples such as cell gene drugs and the like by creatively adopting a micro-fluidic chip technology, intensively completing the enrichment, activation and staining of microorganisms such as bacteria, fungi and the like in complex samples in the micro-fluidic chip and by a biochemical luminescence and high-resolution optical microscopic morphology detection technical method. The method can be suitable for the rapid microbial detection of various complex samples, and the detection method can be successfully applied to the detection and quality control of cell gene drugs and can also be applied to the aseptic detection of biological pharmacy, food industry and environmental detection; the method completes all detection operations in the microfluidic chip, can meet the requirements of processing and detecting low-volume samples and trace samples, and is a totally-enclosed system to avoid secondary pollution.
Description
Technical Field
The invention belongs to the technical field of rapid sterile microorganism detection, and particularly relates to a micro-fluidic chip and a micro-fluidic method for microorganism sorting.
Background
The existing rapid microorganism detection system based on the solid-phase cell counting method is a CHEMINNEX ScanRDI system of the French Mei Liai company. The ScanRDI system is operated by a professional technician, samples are filtered to intercept microorganisms, then dyeing marking is carried out, and counting detection is carried out by adopting a laser scanning technology. The existing ScanRDI system of the solid phase counting product can only complete the detection of simple samples (the simple samples generally refer to the content of small biological molecules or part of water-soluble biological macromolecules, such as nasal drops, injection and the like; the corresponding complex samples generally comprise biological macromolecules of protein, nucleic acid and the like, gene therapy related carriers, cell accessories, such as extracellular vesicles and the like, cell medicines and the like); the existing solid-phase counting product ScanRDI system needs technical personnel for professional sterile detection operation, and the open operation has the possibility of secondary pollution; the existing ScanRDI system of the solid-phase counting product is not suitable for processing trace detection samples of low-volume samples; the existing ScanRDI system of the solid-phase counting product needs technical personnel of professional sterile detection operation to finish manual interpretation after all detection is finished;
disclosure of Invention
The invention aims to provide a micro-fluidic chip and a micro-fluidic method for sorting microorganisms, which can meet the requirements of quick aseptic detection in quality detection and quality control of cell gene drugs and other biological preparations.
Compared with traditional medicines, the cell therapy product has a plurality of specificities in quality attribute and sterile production process: the product has small production batch (only a few to more than ten products are produced in each batch), short effective period (6-8 hours), uniqueness (one dose and one batch) in each product, and no filter sterilization or other sterilization methods can be used in the production process. In the real-time control and the quick release of finished products of the innovative pharmaceutical process, the detection time of the traditional sterile inspection method (culture method) in Chinese pharmacopoeia needs 12 to 14 days, even longer time, and is obviously not suitable.
The product creatively adopts the micro-fluidic chip technology, the enrichment, the activation and the dyeing of microorganisms such as bacteria, fungi and the like in a complex sample are integrally completed in the micro-fluidic chip, and the purpose of quickly and aseptically detecting special biological product samples such as cell gene drugs and the like is realized through biochemical luminescence and a high-resolution optical microscopic morphology detection technical method.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a microfluidic chip for sorting microorganisms, comprising: the chip is divided into an upper chip and a lower chip, a sample inlet and a reagent inlet are oppositely arranged on the upper chip, the sample inlet is connected with a sample linear flow channel, the reagent inlet is connected with a reagent linear flow channel, the sample linear flow channel and the reagent linear flow channel are arranged at a certain angle with the straight line where the sample inlet and the reagent inlet are located, the sample linear flow channel is connected with an S-shaped flow channel after being intersected with the reagent linear flow channel, the other end of the S-shaped flow channel is divided into two rear-end linear flow channels, and the outlet of the rear-end linear flow channel is connected with the inlet of the lower chip; an oil phase inlet is arranged outside the intersection region of the lower chip and the upper chip, a flow channel connected with the oil phase inlet is divided into an upper linear flow channel and a lower linear flow channel, the upper linear flow channel and the lower linear flow channel are connected with two inflow flow channels entering the lower chip from the upper chip, the upper linear flow channel and the lower linear flow channel are divided into rectangular flow channels at the connection position of the inlets of the upper chip and the lower chip, one side of each rectangular flow channel is connected with the inflow flow channel in a cross manner to form a cross focusing hole, the flow channel at the rear end of the cross is connected with a liquid drop enrichment region, the liquid drop enrichment region is in a hexagonal structure, the other end of the liquid drop enrichment region is connected with a buffer region through the inflow flow channel, the buffer region is in a hexagonal structure, the outflow flow channel at the other end of the buffer region is connected with a detection region after being intersected, and the outflow flow channel of the detection region is connected with a waste liquid outlet arranged on the lower chip;
the method is characterized in that: the cross-sectional area of the cross focusing hole is 0.5-2% of the cross-sectional area of the inflow runner.
Preferably, the chip body is made of at least one of silicon chip, PDMS and organic glass.
Preferably, a sample to be detected, an auxiliary reagent and an oil phase reagent are connected to the microfluidic chip and are detected by a high-resolution optical detection system and fluorescence identification sorting.
Preferably, the sample to be tested and/or the oil phase reagent are filtered through a filter. The sample to be tested is filtered to remove impurities except bacteria and retain the bacteria in the sample. The filter membrane used by the invention is suitable for effectively intercepting cells in a sample to be detected and allowing bacteria to pass through without loss.
More preferably, the filter contains a cellulose filter membrane or a modified cellulose filter membrane, the modified cellulose filter membrane is obtained by assembling the cellulose filter membrane through a modified solution containing modified chitosan and modified montmorillonite and a sodium alginate solution layer, the modified chitosan is obtained by modifying chitosan with 3-glycidyloxypropyl trimethoxy silane and/or 2,3-epoxypropyl trimethyl ammonium bromide, and the modified montmorillonite is obtained by modifying lauryl sodium sulfate or a silane coupling agent KH 550. According to the invention, the cellulose filter membrane is assembled and modified, an assembly modified layer is formed on the surface of the cellulose filter membrane, the flow rate and the compression resistance of the cellulose filter membrane can be improved, the tensile strength of the cellulose filter membrane can also be improved, and the common modification of the 3-glycidyl ether oxypropyl trimethoxy silane and the 2,3-epoxypropyl trimethyl ammonium bromide is better than the modification only by adopting the 3-glycidyl ether oxypropyl trimethoxy silane.
Even more preferably, 3-glycidoxypropyltrimethoxysilane is used in an amount of 10 to 40% by weight based on the weight of the chitosan.
Even more preferably, the modified chitosan is obtained by modifying chitosan with 3-glycidoxypropyltrimethoxysilane and 2,3-epoxypropyltrimethylammonium bromide, and the amount of 2,3-epoxypropyltrimethylammonium bromide is 20-41.67wt% of 3-glycidoxypropyltrimethoxysilane.
Still more preferably, the montmorillonite is a calcium-based montmorillonite.
Still more preferably, the silane coupling agent KH550 is used in an amount of 20-60wt% of the calcium-based montmorillonite.
More preferably, the modified montmorillonite is used in an amount of 30-60wt% of the modified chitosan.
The invention discloses application of a modified cellulose filter membrane in microorganism separation in a microfluidic chip, wherein the modified cellulose filter membrane is obtained by assembling a cellulose filter membrane through a modified solution containing modified chitosan and modified montmorillonite and a sodium alginate solution layer, the modified chitosan is obtained by modifying chitosan through 3-glycidyl ether oxypropyl trimethoxy silane and/or 2,3-epoxypropyl trimethyl ammonium bromide, and the modified montmorillonite is obtained by modifying lauryl sodium sulfate or a silane coupling agent KH 550.
Preferably, the modified liquid is formed by mixing modified montmorillonite suspension and modified chitosan solution, and the use amount of the modified montmorillonite is 30-60wt% of the modified chitosan.
The invention discloses a preparation method of a modified cellulose filter membrane, which comprises the steps of preparing modified chitosan, preparing modified montmorillonite, preparing a modified solution, preparing a sodium alginate solution and preparing the modified cellulose filter membrane.
Preferably, in the preparation of the modified chitosan, adding chitosan into an acetic acid solution, adjusting the pH value to 9-10, standing for 4-12h at 20-40 ℃ after the chitosan is separated out, carrying out suction filtration, adding into isopropanol, stirring and dispersing, adding a 3-glycidyl ether oxypropyl trimethoxysilane solution at 70-90 ℃, reacting for 3-12h, after the reaction is finished, carrying out ethanol solution filtration, precipitating the filtrate by using acetone, combining precipitates, and carrying out dialysis treatment for 2-7d to obtain the modified chitosan.
More preferably, in the preparation of the modified chitosan, the acetic acid solution contains 1-3wt% of acetic acid, the chitosan is used in an amount of 1-4wt% of the acetic acid solution, and the chitosan is used in an amount of 10-30wt% of the isopropanol.
More preferably, in the preparation of the modified chitosan, the 3-glycidoxypropyltrimethoxysilane solution is prepared by adding 3-glycidoxypropyltrimethoxysilane to isopropanol, and the 3-glycidoxypropyltrimethoxysilane solution contains 5-20wt% of 3-glycidoxypropyltrimethoxysilane.
More preferably, in the preparation of the modified chitosan, 3-glycidoxypropyltrimethoxysilane is used in an amount of 10 to 40% by weight based on the amount of 3-glycidoxypropyltrimethoxysilane used in the solution.
More preferably, 2,3-epoxypropyltrimethylammonium bromide is also added in the preparation of the modified chitosan, so that the 3-glycidoxypropyltrimethoxysilane solution is prepared by adding 3-glycidoxypropyltrimethoxysilane and 2,3-epoxypropyltrimethylammonium bromide into isopropanol, wherein the 3-glycidoxypropyltrimethoxysilane solution contains 5-20wt% of 3-glycidoxypropyltrimethoxysilane, and the 3-glycidoxypropyltrimethoxysilane solution contains 4-12wt% of 2,3-epoxypropyltrimethylammonium bromide.
Preferably, in the preparation of the modified montmorillonite, calcium-based montmorillonite is dispersed in deionized water, ultrasonic cleaning is carried out, sodium dodecyl sulfate is added, stirring is carried out at 70-90 ℃, the pH value is adjusted to 6-7, treatment is carried out for 2-8h, after the treatment is finished, filtration is carried out, deionized water washing is carried out, absolute ethyl alcohol washing is carried out, drying and grinding are carried out, and the modified montmorillonite is obtained.
More preferably, in the preparation of the modified montmorillonite, the use amount of the calcium-based montmorillonite is 10-40wt% of deionized water.
More preferably, in the preparation of the modified montmorillonite, the usage amount of the sodium dodecyl sulfate is 20-60wt% of the calcium-based montmorillonite.
More preferably, in the preparation of the modified montmorillonite, sodium dodecyl sulfate can be replaced by a silane coupling agent KH550 and ethanol, and the using amount of the silane coupling agent KH550 is 20-60wt% of the calcium-based montmorillonite. The usage amount of the ethanol is 80-120wt% of the calcium-based montmorillonite. After the montmorillonite is modified by adopting a silane coupling agent KH550, chitosan modified by 3-glycidoxypropyltrimethoxysilane and 2,3-epoxypropyltrimethylammonium bromide is used, so that the flow, the compressive property and the tensile strength of the obtained modified cellulose filter membrane are improved.
Preferably, in the preparation of the modified liquid, modified montmorillonite is added into deionized water to prepare modified montmorillonite suspension, then modified chitosan solution is added, and stirring is carried out for 12-36h at 20-40 ℃ to obtain the modified liquid.
More preferably, in the preparation of the modified liquid, the modified montmorillonite suspension contains 0.03-0.12wt% of modified montmorillonite.
More preferably, in the preparation of the modified liquid, the usage amount of the modified montmorillonite suspension is based on the amount of the modified montmorillonite, the modified chitosan solution is prepared by adding the modified chitosan into deionized water, the usage amount of the modified chitosan solution is based on the modified chitosan, and the usage amount of the modified montmorillonite is 30-60wt% of the modified chitosan.
Preferably, in the preparation of the sodium alginate solution, the sodium alginate is added into deionized water, stirred and dissolved, the pH is adjusted to 4-5, and sodium chloride is added to obtain the sodium alginate solution.
More preferably, in the preparation of the sodium alginate solution, the sodium alginate solution contains 0.05 to 0.4 weight percent of sodium alginate, and the sodium alginate solution contains 0.05 to 0.4 weight percent of sodium chloride.
Preferably, in the preparation of the modified cellulose filter membrane, the cellulose acetate filter membrane is immersed in the modified solution for 10-30min, then taken out and immersed in the sodium chloride solution for 1-3min, then the modified cellulose acetate filter membrane is immersed in the sodium alginate solution for 10-30min, then taken out and immersed in the sodium chloride solution for 1-3min, the immersion of the modified solution and the immersion of the sodium alginate solution are repeated for at least 1 time, and finally the modified cellulose filter membrane is obtained after drying at 70-90 ℃ for 18-36 h.
More preferably, in the preparation of the modified cellulose filter, the pore size of the cellulose acetate filter is 0.22 μm.
More preferably, in the preparation of the modified cellulose filter membrane, the cellulose acetate filter membrane is used in an amount of 10 to 30wt% based on the modified solution.
More preferably, the modified cellulose filter membrane is prepared such that the sodium chloride solution contains 0.05 to 0.4wt% sodium chloride.
More preferably, in the preparation of the modified cellulose filter membrane, the weight of the cellulose acetate filter membrane is changed by being impregnated with the modified solution and being impregnated with the sodium alginate solution, so that the usage amount of the cellulose acetate filter membrane is 10-30wt% of the sodium chloride solution and the usage amount of the cellulose acetate filter membrane is 10-30wt% of the sodium alginate solution based on the cellulose acetate filter membrane before impregnation.
The invention adopts the following mode: 1) Processing the sample: the operations of sample filtration, bacterial activation, bacterial marking, fluorescence scanning, optical microscopy and the like are all integrated into a micro-fluidic chip carrier to be completed. The flow direction and the flow speed of the liquid flow are accurately controlled through the driving unit; the shunting regulation and control of a detection sample, an activating agent, a coloring agent and waste liquid are realized through the special design of a flow channel structure; thereby completing the automatic processing of sample enrichment, activation and dyeing under the totally enclosed condition; 2) Need carry out accurate regulation and control to the microenvironment of the bacterial growth in the micro-fluidic chip as required in sample enrichment, activation and dyeing process: the temperature of the chamber during incubation is 30 +/-0.5 ℃; 3) Scanning line by laser with specific wavelength to find out the position of positive fluorescence and calibrating; then, performing microscopic examination on the positive fluorescence position by using an optical microscope to quickly obtain a digital image of the bacteria morphological structure; 4) Through the data contrast analysis to the bacterium detection information image, realize the accurate discernment and the count statistics to the microorganism. Therefore, the following beneficial effects are achieved: the method can be suitable for rapid microbial detection of various complex samples, and can be successfully applied to detection and quality control of cell gene drugs and sterile detection of biological pharmacy, food industry and environmental detection; the operation is simple, and only one-step sample adding is needed; the whole detection operation is completed in the micro-fluidic chip, the treatment and detection of low-volume samples and trace samples can be met, and the micro-fluidic chip is a totally-enclosed system, so that secondary pollution is avoided; the sensitivity is high, and the lowest detection limit is 100cfu/ml; the detection speed is high, and the time from the sample addition to the detection completion is controlled to be 4h; and (4) constructing a sample standard database for detection results. And (3) constructing an artificial intelligent identification network such as u-net, mask-RCNN and the like, identifying different types of microorganism bacteria, counting and detecting different types of microorganisms, and avoiding manual interpretation. Therefore, the invention is a micro-fluidic chip and a method for sorting microorganisms, which can meet the requirements of quick aseptic detection in quality detection and quality control of cell gene drugs and other biological preparations.
Drawings
FIG. 1 is a schematic diagram of an upper chip of a microfluidic chip;
FIG. 2 is a schematic diagram of a lower chip of the microfluidic chip;
FIG. 3 is a cross focus hole pattern in the lower chip;
FIG. 4 is a schematic view of a sample introduction of a microfluidic chip;
FIG. 5 is a cellulose membrane flow chart;
FIG. 6 is a graph showing the rate of change of flow after the modified cellulose filter membrane is subjected to compression treatment;
FIG. 7 is a graph of the tensile strength of a modified cellulose filter membrane.
Reference numerals: 1-upper chip; 11-sample inlet; 12-sample linear flow path; 13-reagent straight flow channel; 14-a reagent inlet; 15-an outlet of the rear end linear flow channel; 16-rear end straight flow channel; the 17-S type flow passages are connected; 2-lower chip; 21-a buffer area; 22-a waste liquid outlet; 23-a flow channel of the detection zone; 24-oil phase inlet; 25-upper straight flow channel; 26-inlet of lower chip; 261-an inflow channel; 27-a droplet enrichment zone; 271-cross rear flow channel; 28-cross focus aperture; 29-lower straight runner; 31-a reagent; 32-oil phase; 33-liquid to be detected; 34-filter.
Detailed Description
The technical scheme of the invention is further described in detail by combining the detailed description and the attached drawings:
example 1:
a method for preparing a modified cellulose acetate filter membrane,
preparing modified chitosan: adding chitosan into acetic acid solution, adjusting pH to 9, standing at 30 ℃ for 8h after chitosan is separated out, carrying out suction filtration, adding into isopropanol, stirring for dispersion, adding 3-glycidyl ether oxypropyl trimethoxysilane solution at 80 ℃, reacting for 6h, after the reaction is finished, carrying out ethanol solution filtration, precipitating filtrate by using acetone, combining precipitates, and carrying out dialysis treatment for 3d to obtain the modified chitosan. The acetic acid solution contains 2wt% of acetic acid, the chitosan is used in an amount of 3wt% of the acetic acid solution, the chitosan is used in an amount of 20wt% of isopropanol, the 3-glycidoxypropyltrimethoxysilane solution is prepared by adding 3-glycidoxypropyltrimethoxysilane to isopropanol, the 3-glycidoxypropyltrimethoxysilane solution contains 10wt% of 3-glycidoxypropyltrimethoxysilane, the 3-glycidoxypropyltrimethoxysilane solution is used in an amount of 20wt% of the chitosan based on the amount of the 3-glycidoxypropyltrimethoxysilane.
Preparing modified montmorillonite: dispersing calcium-based montmorillonite in deionized water, ultrasonically cleaning, adding sodium dodecyl sulfate, stirring at 80 ℃, adjusting the pH to 6, treating for 5 hours, filtering after the treatment is finished, washing with deionized water, washing with absolute ethyl alcohol, drying, and grinding to obtain the modified montmorillonite. The usage amount of the calcium-based montmorillonite is 30wt% of the deionized water, and the usage amount of the sodium dodecyl sulfate is 40wt% of the calcium-based montmorillonite.
Preparing a modified solution: adding modified montmorillonite into deionized water to prepare modified montmorillonite suspension, adding modified chitosan solution, and stirring at 30 deg.C for 24 hr to obtain modified solution. The modified montmorillonite suspension contains 0.05wt% of modified montmorillonite, the using amount of the modified montmorillonite suspension is based on the amount of the modified montmorillonite, the modified chitosan solution is prepared by adding modified chitosan into deionized water, the using amount of the modified chitosan solution is based on the modified chitosan, and the using amount of the modified montmorillonite is 50wt% of the modified chitosan.
Preparation of sodium alginate solution: adding sodium alginate into deionized water, stirring to dissolve, adjusting pH to 4, and adding sodium chloride to obtain sodium alginate solution. The sodium alginate solution contains 0.1wt% of sodium alginate and 0.1wt% of sodium chloride.
Preparing a modified cellulose filter membrane: immersing the cellulose acetate filter membrane into the modified solution for 20min, taking out the cellulose acetate filter membrane, immersing the cellulose acetate filter membrane in a sodium chloride solution for 2min, immersing the modified cellulose acetate filter membrane into a sodium alginate solution for 20min, taking out the cellulose acetate filter membrane, immersing the cellulose acetate filter membrane in the sodium chloride solution for 2min, repeating the immersion of the modified solution and the immersion of the sodium alginate solution for 1 time, and finally drying the cellulose acetate filter membrane for 24h at 80 ℃ to obtain the modified cellulose filter membrane. The pore size of the cellulose acetate filter was 0.22. Mu.m. The usage amount of the cellulose acetate filter membrane is 20wt% of the modified solution, the sodium chloride solution contains 0.1wt% of sodium chloride, and the weight of the cellulose acetate filter membrane is changed by impregnation with the modified solution and impregnation with the sodium alginate solution, so that the usage amount of the cellulose acetate filter membrane is 20wt% of the sodium chloride solution and the usage amount of the cellulose acetate filter membrane is 20wt% of the sodium alginate solution by taking the cellulose acetate filter membrane before impregnation as a reference.
In the case of the example 2, the following examples are given,
the difference between the preparation method of the modified cellulose acetate filter membrane and the embodiment 1 is the preparation of modified chitosan.
Preparing modified chitosan: adding chitosan into acetic acid solution, adjusting the pH value to 9, standing at 30 ℃ for 8 hours after chitosan is separated out, performing suction filtration, adding into isopropanol, stirring for dispersion, adding 3-glycidyl ether oxypropyl trimethoxy silane solution at 80 ℃, reacting for 6 hours, after the reaction is completed, performing ethanol solution filtration, precipitating filtrate by using acetone, combining precipitates, and performing dialysis treatment for 3 days to obtain the modified chitosan. The acetic acid solution contains 2wt% of acetic acid, the chitosan is used in an amount of 3wt% of the acetic acid solution, the chitosan is used in an amount of 20wt% of isopropanol, the 3-glycidyloxypropyltrimethoxysilane solution is prepared by adding 3-glycidyloxypropyltrimethoxysilane and 2,3-epoxypropyltrimethylammonium bromide into isopropanol, the 3-glycidyloxypropyltrimethoxysilane solution contains 10wt% of 3-glycidyloxypropyltrimethoxysilane, the 3-glycidyloxypropyltrimethoxysilane solution contains 8wt% of 2,3-epoxypropyltrimethylammonium bromide, and the 3-glycidyloxypropyltrimethoxysilane solution is used in an amount of 20wt% of the chitosan based on the amount of the 3-glycidyloxypropyltrimethoxysilane.
In the case of the example 3, the following examples are given,
the difference of the preparation method of the modified cellulose acetate filter membrane in the embodiment compared with the embodiment 1 is the preparation of modified montmorillonite.
Preparing modified montmorillonite: dispersing calcium-based montmorillonite in deionized water, ultrasonically cleaning, adding a silane coupling agent KH550 and ethanol, stirring at 80 ℃, adjusting the pH to 6, treating for 5 hours, filtering after the treatment is finished, washing with deionized water, washing with absolute ethanol, drying, and grinding to obtain the modified montmorillonite. The using amount of the calcium-based montmorillonite is 30wt% of deionized water, the using amount of the silane coupling agent KH550 is 40wt% of the calcium-based montmorillonite, and the using amount of the ethanol is 100wt% of the calcium-based montmorillonite.
In the case of the example 4, the following examples are given,
the difference between the embodiment and the embodiment 2 is the preparation of modified montmorillonite.
Preparing modified montmorillonite: dispersing calcium-based montmorillonite in deionized water, ultrasonically cleaning, adding a silane coupling agent KH550 and ethanol, stirring at 80 ℃, adjusting the pH to 6, treating for 5 hours, filtering after the treatment is finished, washing with deionized water, washing with absolute ethanol, drying, and grinding to obtain the modified montmorillonite. The using amount of the calcium-based montmorillonite is 30wt% of deionized water, the using amount of the silane coupling agent KH550 is 40wt% of the calcium-based montmorillonite, and the using amount of the ethanol is 100wt% of the calcium-based montmorillonite.
Example 5:
as shown in fig. 1 to 3, a microfluidic chip for sorting microorganisms has a two-layer structure, and the upper layer is smaller than the lower layer. The flow channel 12 is connected, the reagent inlet 14 is connected with the reagent linear flow channel 13, the sample linear flow channel 12 and the reagent linear flow channel 13 are arranged at a certain angle with the straight lines of the sample inlet 11 and the reagent inlet 14, the sample linear flow channel 12 and the reagent linear flow channel 13 are connected with the S-shaped flow channel 17 after being intersected, the other end of the S-shaped flow channel 17 is divided into two rear-end linear flow channels 16, and the outlet 15 of the rear-end linear flow channel 16 is connected with the inlet 26 of the lower chip; an oil phase inlet 24 is arranged outside the intersection region of the lower chip 2 and the upper chip 1, a flow channel connected with the oil phase inlet 24 is divided into an upper linear flow channel 25 and a lower linear flow channel 29, the upper linear flow channel 25 and the lower linear flow channel 29 are connected with two inflow flow channels 261 entering the lower chip from the upper chip, the upper linear flow channel 25 and the lower linear flow channel 29 are divided into rectangular flow channels at the connection position of the inlets of the upper chip and the lower chip, one side of the rectangular flow channel is connected with the inflow flow channel 261 in a cross manner to form a cross focusing hole 28, the cross rear end flow channel 271 is connected with a liquid drop enrichment area 27, the liquid drop enrichment area 27 is in a hexagonal structure, the other end of the liquid drop enrichment area 27 is connected with a buffer area 21 through the inflow flow channel, the buffer area 21 is in a hexagonal structure, the outflow flow channel at the other end of the buffer area 21 is connected with a detection area after intersecting, and the outflow flow channel of the detection area is connected with a waste liquid outlet 22 arranged on the lower chip.
The upper chip structure: 40mm by 42.5mm by 1.5mm; lower chip architecture: 60mm by 80mm by 1.5mm.
The aperture of the sample inlet, the reagent inlet, the outlet of the rear end linear flow channel, the inlet of the lower chip, the oil phase inlet and the waste liquid outlet is 1.6mm.
The width of the basic runner is 0.2mm, and the height is 0.2mm. The flow channels which are not suitable for the parameters of the basic flow channel are specially explained, and the flow channels which are not specially explained are all suitable for the parameters of the basic flow channel.
The width of the flow channel forming the cross connection is 0.2mm, the height is 0.03mm, and the aperture of the cross focus is 0.02mm. The flow channels connected with the turning parts of the cross-shaped connected flow channels are basic flow channels and are suitable for the width of the basic flow channels.
The width of an inlet of the enrichment region is 0.03mm, and the height of the enrichment region is 0.03mm; the enrichment zone was 30mm long, 25mm wide, 1.3mm high and had a volume of about 2mL. The flow channel behind the liquid drop enrichment area is suitable for parameters with the width of 0.03mm and the height of 0.03 mm.
The buffer area has a side length of 5mm and a height of 1.3mm.
The aperture of the flow channel in the detection area is 0.01mm.
The chip main body is made of a silicon wafer.
Example 6:
as shown in fig. 4, a method for sorting microorganisms includes the microfluidic chip of example 5.
In order to avoid air bubbles from entering to influence the filtering and reagent processing effects, the liquid inlet hole is pre-filled with pre-filling liquid such as washing liquid/buffer solution before the sample is added, and the process is controlled and driven by the liquid control processing unit. The pipeline is communicated with the liquid inlet of the chip in an external steel needle/PTFE catheter mode, the sealing performance can be ensured, meanwhile, the reagent pipeline is connected with a 0.22 mu m filter, and meanwhile, the reagent pipeline and the waste liquid pipeline are provided with bubble sensing/volume recording sensing devices. The Polytetrafluoroethylene (PTFE) tubes are connected by a connector, the connector is a luer connector, and the corresponding reagent and the sample are connected into the inlet port by the luer connector. A0.22 μm filter was placed at the luer fitting, and the filter membrane in the filter was the modified cellulose acetate filter membrane prepared in example 1.
By adopting a droplet microfluidic technology, introducing an oil phase reagent into a pre-filled chip by using a micro-injection pump or a pressure pump, then introducing a sample to be detected and a dyeing reagent to realize water-in-oil type microbial unicell package, and then carrying out fluorescence identification sorting detection by using a high-resolution optical detection system.
The sample liquid is injected into the chip through a sample inlet on the upper chip, the dye is injected into the chip through a reagent inlet, the sample liquid and the dye are mixed at the intersection of the pipelines, the mixture is uniformly mixed in the flowing process of the S-shaped pipeline, and then the mixture is divided into two parts to enter the lower layer and flow to the liquid drop generating position. Meanwhile, the oil phase enters the chip through the oil phase inlet of the lower chip and is also divided into two parts through the pipeline, and the sample liquid is continuously sheared into liquid drops at the liquid drop generation part due to shearing force. The generated liquid drops enter the enrichment area through a pipeline, and because the density of the sample is smaller than that of the oil phase, the liquid drops are gathered at the top of the flow channel in the enrichment area and are densely arranged, so that the enrichment effect is achieved, and the oil phase passes through the bottom of the enrichment area. The enrichment region can enrich liquid drops with the volume of about 2ml, and is followed by a buffer region, so that redundant liquid drops in the enrichment region can be prevented from escaping.
After the sample is completely incubated, the chip is inverted, and due to the influence of density, the liquid drops move upwards and move along the pipeline to the detection area under the pushing of the oil phase. The detection area only allows one liquid drop to pass through at one time, the liquid drop is compressed and deformed due to the fact that the pipeline is narrow, the contact area of the laser is increased, the fluorescent signal is detected, the signal is output, and finally the liquid drop is discharged through the chip outlet and used for follow-up operation.
The invention can adopt PC and MCU and various control components to realize automatic control.
Example 7:
a method for sorting microorganisms, which is a difference from example 6 in that the filter membrane in the filter is the modified cellulose acetate filter membrane prepared in example 2.
Example 8:
a method for sorting microorganisms, which is a difference from example 6 in that the filter membrane in the filter is the modified cellulose acetate filter membrane prepared in example 3.
Example 9:
a method for sorting microorganisms, which is a difference from example 6 in that the filter membrane in the filter is the modified cellulose acetate filter membrane prepared in example 4.
Test example:
1. flow measurement
Test samples: examples 1-4 the resulting modified cellulose acetate filters were prepared.
And (3) testing the flow of the test sample by adopting a dead-end filtration method, recording the time consumed by filtering 100mL of pure water by using a filter membrane under-0.07 MPa, and calculating the flow according to the area of the test sample.
The flow performance of the modified cellulose acetate filter membrane obtained by the method of the embodiment is tested, and the result is shown in fig. 5, wherein S1 is embodiment 1, S2 is embodiment 2, S3 is embodiment 3, and S4 is embodiment 4, the method comprises the steps of firstly modifying chitosan by using 3-glycidyl ether oxypropyltrimethoxysilane to obtain modified chitosan, then adding calcium-based montmorillonite into a sodium dodecyl sulfate solution for modification to obtain modified montmorillonite, then compounding the modified chitosan and the modified montmorillonite into a modified solution, then adding the cellulose acetate filter membrane into the modified solution for treatment, then treating in a sodium alginate solution for layer assembly, and at least circularly assembling the layers twice to obtain the modified cellulose acetate filter membrane; furthermore, 2,3-epoxypropyl trimethyl ammonium bromide can be added in the modification of the chitosan to jointly modify the chitosan, and after the chitosan is jointly modified by 3-glycidyl ether oxypropyl trimethoxy silane and 2,3-epoxypropyl trimethyl ammonium bromide, the modified cellulose acetate filter membrane prepared by the chitosan has higher flow, which shows that the effect of improving the flow performance of the modified cellulose acetate filter membrane is realized by the modification of the chitosan; after 2,3-epoxypropyl trimethyl ammonium bromide is used for modifying chitosan, montmorillonite is modified by silane coupling agent KH550, the flow change of the prepared modified cellulose acetate filter membrane is not obvious, and when 3-glycidoxypropyltrimethoxysilane and 2,3-epoxypropyl trimethyl ammonium bromide are used for jointly modifying chitosan, the silane coupling agent KH550 modified montmorillonite and the modified chitosan are used together, so that the flow of the obtained modified cellulose acetate filter membrane is improved.
2. Test of compression resistance
Test samples: examples 1-4 the resulting modified cellulose acetate filters were prepared.
And pre-pressing the test sample for 0.5h by adopting the pressure of 0.2MPa, and then testing the flow of the pre-pressed test sample according to a flow testing method. And calculating the flow rate change rate.
The invention tests the compression resistance of the modified cellulose acetate filter membrane obtained by the method of the embodiment, and the modified cellulose acetate filter membrane is characterized by the flow rate change, wherein the positive value is positive improvement, and the negative value is negative reduction, and the result is shown in figure 6, wherein S1 is embodiment 1, S2 is embodiment 2, S3 is embodiment 3, and S4 is embodiment 4, the invention firstly modifies chitosan by adopting 3-glycidol ether oxypropyltrimethoxysilane to obtain modified chitosan, then adds calcium-based montmorillonite into sodium dodecyl sulfate solution to modify to obtain modified montmorillonite, then compounds the modified chitosan and the modified montmorillonite into modified solution, then adds the cellulose acetate filter membrane into the modified solution to treat, then treats in the sodium alginate solution to carry out layer assembly, and at least recycles layer assembly twice to obtain the modified cellulose acetate filter membrane; furthermore, 2,3-epoxypropyl trimethyl ammonium bromide can be added in the modification of the chitosan to jointly modify the chitosan, and after the chitosan is jointly modified by 3-glycidoxypropyltrimethoxysilane and 2,3-epoxypropyl trimethyl ammonium bromide, the modified cellulose acetate filter membrane prepared from the chitosan has higher flow change rate, which indicates that the modification of the chitosan has the effect of improving the flow change rate of the modified cellulose acetate filter membrane; after 2,3-epoxypropyl trimethyl ammonium bromide is used for modifying chitosan, montmorillonite is modified by silane coupling agent KH550, the flow rate change rate of the prepared modified cellulose acetate filter membrane is improved, and when 3-glycidoxypropyltrimethoxysilane and 2,3-epoxypropyl trimethyl ammonium bromide are used for jointly modifying chitosan, the silane coupling agent KH550 modified montmorillonite and the modified chitosan are used together, and the flow rate of the obtained modified cellulose acetate filter membrane is obviously improved.
3. Mechanical Property test
Test samples: the modified cellulose acetate filters prepared in examples 1 to 4 were cut into a shape of 10X 100 mm.
The test sample is kept stand for 24 hours at 25 ℃ and 50% humidity, and then the sample is tested by a tensile tester.
The tensile property of the modified cellulose acetate filter membrane obtained by the method of the embodiment is tested, and the result is shown in fig. 7, wherein S1 is embodiment 1, S2 is embodiment 2, S3 is embodiment 3, and S4 is embodiment 4, the invention firstly modifies chitosan by using 3-glycidyl ether oxypropyltrimethoxysilane to obtain modified chitosan, then adds calcium-based montmorillonite into a sodium dodecyl sulfate solution for modification to obtain modified montmorillonite, then compounds the modified chitosan and the modified montmorillonite into a modified solution, then adds the cellulose acetate filter membrane into the modified solution for treatment, then treats the modified chitosan filter membrane in a sodium alginate solution for layer assembly, and at least recycles the layer assembly twice to obtain the modified cellulose acetate filter membrane; furthermore, 2,3-epoxypropyl trimethyl ammonium bromide can be added in the modification of the chitosan to jointly modify the chitosan, and after the chitosan is jointly modified by 3-glycidyl ether oxypropyl trimethoxy silane and 2,3-epoxypropyl trimethyl ammonium bromide, the modified cellulose acetate filter membrane prepared by the chitosan has higher tensile strength, which shows that the effect of improving the tensile strength of the modified cellulose acetate filter membrane is realized through the modification of the chitosan; after 2,3-epoxypropyl trimethyl ammonium bromide is used for modifying chitosan, montmorillonite is modified by silane coupling agent KH550, the tensile strength of the prepared modified cellulose acetate filter membrane is improved, and when 3-glycidoxypropyltrimethoxysilane and 2,3-epoxypropyl trimethyl ammonium bromide are used for jointly modifying chitosan, the silane coupling agent KH550 modified montmorillonite and the modified chitosan are used together, so that the tensile strength of the obtained modified cellulose acetate filter membrane is obviously improved.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (10)
1. A microfluidic chip for sorting microorganisms, comprising: the chip is divided into an upper chip (1) and a lower chip (2), a sample inlet (11) and a reagent inlet (14) are oppositely arranged on the upper chip (1), the sample inlet (11) is connected with a sample linear flow channel (12), the reagent inlet (14) is connected with a reagent linear flow channel (13), the sample linear flow channel (12) is connected with an S-shaped flow channel (17) after being intersected with the reagent linear flow channel (13), the other end of the S-shaped flow channel (17) is divided into two rear-end linear flow channels (16), and an outlet (15) of the rear-end linear flow channel (16) is connected with an inlet (26) of the lower chip; an oil phase inlet (24) is arranged outside the intersection region of a lower chip (2) and an upper chip (1), a flow channel connected with the oil phase inlet (24) is divided into an upper linear flow channel (25) and a lower linear flow channel (29), the upper linear flow channel (25) and the lower linear flow channel (29) are connected with two inflow flow channels (261) entering the lower chip from the upper chip, the upper linear flow channel (25) and the lower linear flow channel (29) are divided into rectangular flow channels at the connection positions of the inlets of the upper chip and the lower chip, one side of each rectangular flow channel is connected with the inflow flow channel (261) in a cross manner to form a cross focusing hole (28), the cross rear end flow channel (271) is connected with a liquid drop enrichment area (27), the liquid drop enrichment area (27) is of a hexagonal structure, the other end of the liquid drop enrichment area (27) is connected with a buffer area (21) through the inflow flow channel, the buffer area (21) is of a hexagonal structure, the outflow flow channel at the other end of the buffer area (21) is connected with a waste liquid waste outlet (22) arranged on the lower chip after intersection, and the outflow channel of the detection area is connected with the waste liquid waste outlet (22) arranged on the lower chip;
the method is characterized in that: the cross-sectional area of the cross focusing hole (28) is 0.5-2% of the cross-sectional area of the inflow channel (261).
2. A method of sorting microorganisms on a microfluidic chip, comprising: the microfluidic chip comprises the microfluidic chip of claim 1, wherein a sample to be detected, an auxiliary reagent and an oil phase reagent are connected onto the microfluidic chip and are detected by a high-resolution optical detection system and fluorescence identification sorting.
3. The microfluidic chip based method for sorting microorganisms according to claim 2, wherein: the sample to be tested and/or the oil phase reagent are filtered by a filter (34).
4. The microfluidic chip based method for sorting microorganisms according to claim 3, wherein: the filter (34) contains a cellulose filter membrane or a modified cellulose filter membrane, the modified cellulose filter membrane is obtained by assembling a cellulose filter membrane with a modified solution containing modified chitosan and modified montmorillonite and a sodium alginate solution layer, the modified chitosan is obtained by modifying chitosan with 3-glycidyl ether oxypropyl trimethoxy silane and/or 2,3-epoxypropyl trimethyl ammonium bromide, and the modified montmorillonite is obtained by modifying with sodium dodecyl sulfate or a silane coupling agent KH 550.
5. The microfluidic chip based method for sorting microorganisms according to claim 4, wherein: the usage amount of the 3-glycidoxypropyltrimethoxysilane is 10-40wt% of the chitosan.
6. The microfluidic chip based method for sorting microorganisms according to claim 4, wherein: the modified chitosan is obtained by modifying chitosan with 3-glycidoxy propyl trimethoxy silane and 2,3-epoxypropyl trimethyl ammonium bromide, and the amount of 2,3-epoxypropyl trimethyl ammonium bromide is 20-41.67wt% of 3-glycidoxy propyl trimethoxy silane.
7. The microfluidic chip based method for sorting microorganisms according to claim 4, wherein: the montmorillonite is calcium-based montmorillonite.
8. The microfluidic chip based method for sorting microorganisms according to claim 4, wherein: the usage amount of the silane coupling agent KH550 is 20-60wt% of the calcium-based montmorillonite.
9. The use of a modified cellulose filter membrane in the sorting of microorganisms in the microfluidic chip according to claim 1, wherein the modified cellulose filter membrane is obtained by assembling a cellulose filter membrane with a modified solution containing modified chitosan and modified montmorillonite and a sodium alginate solution layer, the modified chitosan is obtained by modifying chitosan with 3-glycidyl ether oxypropyltrimethoxysilane and/or 2,3-epoxypropyltrimethylammonium bromide, and the modified montmorillonite is obtained by modifying sodium dodecyl sulfate or a silane coupling agent KH 550.
10. The use of a modified cellulose filter according to claim 9 in the sorting of microorganisms in a microfluidic chip, wherein: the modified liquid is formed by mixing modified montmorillonite suspension and modified chitosan solution, and the use amount of the modified montmorillonite is 30-60wt% of the modified chitosan.
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